ABSTRACT Our long term goal is to elucidate the mechanisms responsible for the functional polarity and retinal- support functions of the retinal pigment epithelium (RPE). The vast array of support functions the RPE performs for the neural retina require RPE-specific polarity of solute transporters, channels and nutrient receptors and functional tight junctions. The polarity of RPE transporters is frequently opposite to the polarity displayed by the same transporters in other body epithelia. Our central hypothesis is that the organization of RPE polarity and its selective blood-retinal barrier properties is dictated by a tissue-specific configuration of the trafficking machinery in RPE cells and by instructive interactions from choroid endothelial cells. Indeed, we recently reported that RPE cells lack a key clathrin adaptor, AP1B, that sorts basolateral PM proteins in most body epithelia, which explains the reversed apical polarity or non-polarized distribution of several RPE PM proteins (e.g. the coxsackie adenovirus receptor (CAR) and neural adhesion molecule (NCAM)). On the other hand, the mechanisms that regulate the reversed apical polarity of some basolateral PM protein in RPE, e.g. the chloride channel ClC-2 and Na,K-ATPase is not explained by lack of this clathrin adaptor. The research plan of this proposal is focused on understanding the sorting mechanisms and functions of proteins involved in the transport of fluid by RPE, a process of major significance in RPE physiology and retinal pathology, and on characterizing a novel choroid-supported mechanism for the assembly of functional RPE tight junctions. Specific aim 1 will focus on ClC-2, a key basolateral regulator of cell volume in many epithelia which we recently found, unexpectedly, to be localized to the apical PM and primary cilium of RPE. Specific aim 2 will focus on NKCC-1 a key Cl- co-transporter in the apical PM of RPE which is expressed basolaterally in other body epithelia and on Bestrophin, a basolateral chloride channel which was recently crystallized and is now therefore amenable for structure-function sorting studies. Finally, specific aim 3 will characterize an exciting new mechanism we have uncovered that regulates the blood-retinal outer barrier through choroid endothelial signals that regulate the maturation of the Bruch basement membrane. We anticipate that the new information and concepts provided by these studies will contribute important insights on the Physiology and Pathology of the Outer Retina that may help generate novel therapeutic strategies for the treatment of diseases of the outer retina.